丙烯腈致大鼠肝细胞损伤中PI3K/AKT信号通路的作用
Role of PI3K/AKT Signaling in Acrylonitrile-induced Hepatocyte Injury in Rats
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摘要: 为探讨丙烯腈(acrylonitrile, ACN)致大鼠肝细胞损伤中PI3K/AKT信号通路的作用，随机将60只雄性SD大鼠分为5组：对照组，ACN低、中、高剂量染毒组和干预组。分别给予玉米油，11.5 mg·kg-1、23.0 mg·kg-1、46.0 mg·kg-1 ACN和300 mg·kg-1 N-乙酰基半胱氨酸(NAC)+46.0 mg·kg-1 ACN灌胃染毒，灌胃容积为5.0 mL·kg-1，1 次·d-1，6 d·周-1，共28 d。最后一次染毒1 d后，大鼠麻醉心脏采血后处死，取肝脏进行实验。(1)原位末端标记法(TUNEL)观察肝细胞凋亡水平；(2)定量逆转录聚合酶链反应法(qRT-PCR)检测pi3k、akt、bad、bcl-2、cyt c和caspase-3基因表达水平；(3)免疫印迹法(WB)检测PI3K/AKT信号通路和线粒体凋亡通路相关蛋白的表达水平。研究表明：(1)细胞凋亡方面。ACN低、中剂量组凋亡细胞较对照组显著增多(P<0.05)。(2)mRNA相对表达水平方面。与对照组相比，ACN低剂量组bad、caspase-3显著升高(P<0.05)，bcl-2显著降低(P<0.05)；ACN中剂量组pi3k、bcl-2降低(P<0.05)，cyt c升高(P<0.05)；ACN高剂量组pi3k、bcl-2降低，bad升高(P<0.05)；ACN各剂量组akt无显著差异(P>0.05)；NAC组bad较ACN高剂量组降低(P<0.05)。(3)蛋白相对表达水平方面。与对照组相比，ACN低剂量组pro-Caspase-3降低，cleaved-Caspase-3升高(P<0.05)；ACN中剂量组PI3K、p-Bad和pro-Caspase-3降低，Caspase-9、Bad、Bax和Cyt c升高，Bcl-2/Bax值降低(P<0.05)；ACN高剂量组p-PI3K、p-AKT、Bcl-2、p-Bad和pro-Caspase-3降低，Bad、Bax、Caspase-9、Cyt c和cleaved-Caspase-3升高，Bcl-2/Bax值降低(P<0.05)；ACN各剂量组AKT无显著差异(P>0.05)。与ACN高剂量组相比，NAC组PI3K、p-Bad和pro-Caspase-3升高，Bax、Caspase-9和cleaved-Caspase-3降低(P<0.05)。ACN抑制PI3K/AKT信号通路及激活线粒体凋亡途径可能是大鼠肝细胞损伤的原因之一。
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关键词:
- 丙烯腈 /
- 大鼠 /
- 细胞凋亡 /
- PI3K/AKT信号通路 /
- 肝细胞
Abstract: To determine how acrylonitrile (ACN) affected the PI3K/AKT signaling in rat hepatocytes, sixty male Sprague-Dawley rats were randomly divided into 5 groups: control group, ACN low dose group (11.5 mg·kg-1), ACN medium dose group (23.0 mg·kg-1), ACN high dose group (46.0 mg·kg-1) and intervention group by gavage administered with the volume of 5.0 mL·kg-1 once a day for 6 days a week for a total of 28 days, respectively. One day after the last gavage, rats were anesthetized for blood collection and sacrificed, and livers were harvested for additional assays. (1) Terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) was used to detect apoptosis; (2) Quantitative reverse transcription polymerase chain reaction method (qRT-PCR) was employed to examine gene expression of pi3k, akt, bad, bcl-2, cyt c, and caspase-3; (3) Western Blot (WB) was utilized to measure the expression level of protein related to PI3K/AKT signaling pathway and mitochondrial apoptosis pathway. Results indicated: (1) ACN low and medium group present a significant apoptotic phenomenon compared to control group (P<0.05). (2) mRNA level. In the ACN low dose group, bad and caspase-3 were considerably upregulated (P<0.05), while bcl-2 was dramatically downregulated (P<0.05); in ACN medium dose group, pi3k and bcl-2 were decreased (P<0.05), whereas cyt c was increased (P<0.05). In the ACN high-dose group, pi3k and bcl-2 decreased, while bad increased (P<0.05). There was no significant difference in akt between ACN dose groups (P>0.05). bad in N-acetylcysteine (NAC) group was lower than that in ACN high-dose group (P<0.05). (3) Protein level. Compared with the control group, pro-Caspase-3 was decreased and cleaved Caspase-3 was increased in the low-dose ACN group (P<0.05); in ACN medium-dose group, PI3K, p-Bad, pro-Caspase-3 and Bcl-2/Bax were decreased, Caspase-9, Bad, Bax and Cyt c were increased (P<0.05). In the ACN high-dose group, p-PI3K, p-AKT, Bcl-2, p-Bad, pro-Caspase-3 and Bcl-2/Bax were decreased, while Bad, Bax, Caspase-9, Cyt c, cleaved Caspase-3 were increased (P<0.05). There was no significant difference in AKT among the ACN dose groups (P>0.05). Compared with the high-dose ACN group, PI3K, p-Bad and pro-Caspase-3 were increased in the NAC group, while Bax, Caspase-9, and cleaved Caspase-3 were decreased (P<0.05). It is concluded that ACN may induce hepatocyte injury via PI3K/Akt signaling pathway and the activation of mitochondrial apoptosis pathway in rats.-
Key words:
- acrylonitrile /
- rats /
- cell apoptosis /
- PI3K/AKT signaling pathway /
- hepatocyte
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